Two thermophilic bacilli were isolated from mud and water samples of the Ayder and Kestanbol hot springs in the provinces of Rize and Canakkale, respectively, in Turkey. Strains AB04(T) and K4(T) were sporulating, Gram-positive, rod-shaped bacteria. These isolates were moderately thermophilic (with an optimum temperature for growth of 50-55degreesC), facultative anaerobes able to grow on a wide range of carbon sources including D-glucose, D-raffinose, D-sucrose, D-xylose, D-fructose, L-arabinose, maltose, D-mannose and D-mannitol. Analysis of the 16S rRNA gene sequences showed that these isolates resembled Anoxybacillus flavithermus DSM 2641(T) and Anoxybacillus gonensis NCIMB 13933(T). DNA-DNA hybridization data revealed that thermophilic isolate AB04(T) has only 51-20% relatedness to A. flavithermus, 45-1% relatedness to Anoxybacillus pushchinoensis and 68-6% relatedness to A. gonensis. Thermophilic isolate K4(T) showed only 60.4% relatedness to A. flavithermus, 42.9% relatedness to A. pushchinoensis and 38.5% relatedness to A. gonensis. On the basis of the DNA-DNA hybridization data, isolates AB04(T) and K4(T) are not related to A. flavithermus DSM 2641(T), A. pushchinoensis DSM 12423(T) or A. gonensis NCIMB 13933(T) at the species level, but show relatedness to one another of 40.5%. On the basis of the data presented, it is proposed that strains AB04(T) ( = NCIMB 13972(T) = NCCB 100050(T)) and K4(T) ( = NCIMB 13971(T) = NCCB 100051(T)) be designated as the type strains of Anoxybacillus ayderensis sp. nov. and Anoxybacillus kestanbolensis sp. nov., respectively.